Roche 454 GS FLX+

454 Fastq Files (GEO:GSE48032)


Starting Material and Enrichment

The MAQC A and MAQC B RNAs were subjected to two rounds of polyA enrichment at a single laboratory site before distribution to the other 454 data generation core laboratories.  Each RNA sample was enriched using the Oligotex mRNA Mini Kit (Qiagen), starting from 60 µg of RNA, according to manufacturer’s instructions, using the spin column <0.25 mg method.  A second enrichment step was performed following step 5 of the protocol, according to the manufacturer’s instructions.  Final elution was performed twice using 50 µl of 700C OEB elution buffer.  The resulting enriched RNA was quantified by Nanodrop spectrophotometry and evaluated on the Agilent Bioanalyzer 2100 using an RNA PicoChip. 

Library Construction

Library synthesis and sequencing was performed with MAQC A and MAQC B samples at three core laboratory sites.  Each site constructed one cDNA library from each of the polyA-enriched RNA samples.  Enriched RNA (200 ng) was reduced to 19 µl in a vacuum centrifuge at 60 oC, followed by library construction using  the Roche cDNA Rapid Library Preparation Method Manual XL+ (May 2011) with the following modifications: (1) RNA Fragmentation Reagent kit (AM#8740, Life Technologies) was used in place of the RNA Fragmentation Solution; (2) all magnetic particle concentrator (MPC) pelleting steps were held for 2 minutes; (3) Roche rapid library multiplex identifier (RL-MIDs) adaptors were used for the adaptor ligation step; (4) the final libraries were quantified using a Qubit fluorometer (Life Technologies) and average fragment sizes were determined by analyzing 1 µl of the libraries on the Agilent Bioanalyzer 2100 using a High-Sensitivity DNA LabChip; and (5) the library concentrations were determined using the average fragment size from the Bioanalyzer analysis.  Final samples were diluted to 1x108 molecules/µl in Tris-HCl pH 8 buffer with 0.001% Tween-20. 

Template Enrichment and Sequencing

Libraries were diluted to 1x106 molecules/µl for sequencing.  Emulsion-based clonal amplification and sequencing on the Roche 454 Genome Sequencer FLX+ was performed according to the manufacturer’s instructions (454 Life Sciences).  Each library was sequenced on one full PicoTiterPlate (PTP) per laboratory site.  An additional PTP per library was sequenced at one of the laboratories for a total of four PTPs per MAQC sample.  Sequencing was done using the Roche XL+ sequencing kit with software version 2.6 or 2.8 with Flow Pattern A.  Signal processing and base calling were performed using the bundled 454 Data Analysis Software (v.2.6 and 2.8).